Chemotaxis plays a central role in diverse biological processes, including metastasis of cancer cells, inflammatory responses involving movement of neutrophils and macrophage, migration of neural crest cells, embryonic morphogenesis, and aggregation in Dictyostelium. The underlying processes that control chemotaxis in eukaryotic cells are highly conserved between Dictyostelium and man and utilize common factors in integrated circuits to control directional cell movement toward a chemoattractant. The key component of this process is the ability of cells to produce a pseudopod at the leading edge in the direction of the chemoattractant source and contraction of the posterior of the cell. Our goal is to understand the mechanisms by which cells sense the direction of the chemoattractant gradient and utilize downstream signaling pathways to control this reorganization of the cytoskeleton. In this application, we focus on the role of the Dictyostelium PAK/Ste2O family member PAKa, which we have demonstrated is essential for the regulation of myosin assembly during chemotaxis, and the Rac family of small GTPases. We propose to elucidate the mechanisms by which PAKa is activated in response to chemoattractant signaling and understand the molecular interactions that regulate its subcellular localization and function in the posterior of chemotaxing cells. In addition, we propose experiments to examine the mechanism of activation of Rac1, which we have demonstrated is a key player in the control of the actin cytoskeleton during chemotaxis. These studies include investigating the kinetics and regulation of Rac1 activation and determining the possible changes in the subcellular localization of Rac1 in cells responding to a chemoattractant gradient. To understand the upstream regulation of Rac activation, we propose to examine the role of putative Rac exchange factors (GEFs) that control the activation of Rac proteins in response to various cellular stimuli. Using cell biological and molecular genetic approaches in Dictyostelium, we will define the role of Rac GEFs and how they are regulated, with a specific focus on examining changes in their subcellular localization and the role of such changes in regulating directional responses. Our proposed studies should also help elucidate how PAKa and Rac integrate into other signaling pathways that are essential for controlling the ability of Dictyostelium cells to directionally sense and respond to chemoattractant gradients. Understanding the mechanisms that regulate PAKa, Rac1, and RacGEFs in Dictyostelium will help define general mechanisms controlling cell polarization and movement that should be applicable to determining how chemotaxis is regulated in a broad range of cells.